The present invention relates generally to the fields of analytics of proteins, peptides, DNA and RNA. More particularly, the invention concerns determination of the exact ratio of protein, peptide, DNA or RNA isoforms in a sample. More specially, the invention relates to the use of a defined precursor to measure the relative amounts of closely related protein, peptide, DNA or RNA isoforms such as phosphoisoforms from a synthetic or biological sample.
With the completion of the Human Genome Project, the emphasis is shifting to examining the protein complement in the human organism. This has given a rise to the science of proteomics, the study of the whole amount of the proteins produced by a cell type of an organism. At the same time, there has been a revival of interest in proteomics in many prokaryotes and lower eukaryotes as well.
The term proteome refers to all the different proteins expressed by a genome and thus involves the identification of proteins in the body and the detection of their physiological and pathophysiological function. The about 30.000 genes, defined by the Human Genome project translate into 30.000 up to 1 million of proteins, when alternate splicing and post-translational modifications are considered. While a specific genome remains unchanged to a high extent, the proteins in any particular cell change dramatically as genes are turned on and off in response to extracellular stimulation.
As a reflection of the dynamic nature of the proteome, the term “functional proteome” is preferred by some scientists, which describes the whole amount of proteins produced by a single cell in a single time frame. Finally, it is believed, that through proteomics, new disease markers and drug targets can be identified that will help design new products to prevent, diagnose and treat disease.
One of the most common protein modifications is phosphorylation. It is estimated, that about 30% of all proteins in mammalian cells are phosphorylated at any given time and that about 5% of the vertebrate genome encodes protein kinases and phosphatases, underlining the importance of this protein modification. The presence of various protein kinases and phosphatases permits the use of quickly reversible phosphorylation in a vast number of different, highly regulated pathways and functions, including signal transduction, cell division, apoptosis regulation, and cell differentiation.
Knowledge of the phosphorylation site is crucial for detailed understanding various different regulatory processes in cells. This knowledge requires sensitive and highly sophisticated analysis methods. Theoretically, the most sensitive method for detecting of phosphorylation is to incorporate radioactive phosphorus isotopes before phosphopeptide mapping and/or Edman degradation. However, e.g. in tissue samples, the incorporation of radioactive isotopes is not possible or is very inefficient in the cause of cell culture due to the presence of unlabeled ATP. Moreover, high levels of radioactive derivatives, incorporated in the cell may cause cellular damage and can thereby alter phosphorylation.
Protein phosphorylation analysis by mass spectrometry has a structural and two quantitative aspects. The structural aspect refers to the identification of the phosphorylated site (recognition of phosphorylated amino acid residue and its position within the protein chain). The quantitative aspects comprise the determination of the phosphorylation degree (equivalent term: phosphorylation stoichiometry), which is the molar ratio between the phosphorylated and unphosphorylated residue at a particular position in the protein chain (relative quantification), and the determination of the amount of phosphoprotein (or phosphopeptide) present, which carries a particular phosphorylation site (absolute quantification). Considering the fact, that protein phosphorylation analysis is of major interest in many laboratories around the world, it is surprising, that more information about protein phosphorylation sites and about the ratio of phosphorylation of these sites has not been gathered since the discovery of protein phosphorylation. One reason is the generally low phosphorylation stoichiometry of most proteins such that phosphopeptides are widely underrepresented in the generated protein or peptide mixtures.
As the issue of phosphorylation stoichiometry is less commonly addressed, several publications deal with the problem of determining the ratio of two different targets, in particular the ratio of two different target-peptides.
US 2004/0119010 A1 discloses a method for quantifying the amounts of proteins and peptides, including those that are closely related isoforms, using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). According to this laid open publication, a method of quantitatively comparing the amount of a plurality of structurally distinct proteins, or peptides is disclosed. This method comprises the steps of obtaining one or more samples containing said multiply distinct target proteins or peptides, providing a standard, wherein the standard is a derivative of the target protein or peptide of interest at a known or measurable quantity, co-crystallizing the target proteins or the standard with a matrix, analyzing the crystallized target proteins or peptides and standard using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry; and determining relative and absolute protein or peptide analyzed, that is present in the sample. According to the teaching of this document, the standards are proteins or peptides derived or synthesized directly from the proteins of interest, what implicates imprecisions in the exact quantity of the used standards.
A further problem, which has to be mentioned, is the so called response, also called flyability in mass spectrometry of phosphoproteins. H. Steen et al., teaches in: stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS, Steen H., PNAS, 11, 2005, that determination of the stoichiometry of phosphoproteins would be an easy task, if peptides and their phosphorylated cognates had identical ionization/detection efficiencies because it would be sufficient to monitor the peptide and the phosphorylated complement and calculate the intensity ratio between these two species. In this publication, H. Steen suggests to determine the flyability for at least two samples of a peptide/phosphopeptide pair. These two samples must be interconnected by a treatment resulting in partial dephosphorylation (e.g. induced by phosphatase), so that the changes in the phosphorylation degree between these two samples are interconnected in the following way: the decrease in the amount of phosphopeptide in the treated sample is equivalent to the increase of the nonphosphorylated cognate in that sample. To test this approach, several synthetic peptides and their phosphorylated cognates were purified to homogeneity and quantified by amino acid analysis. These synthetic standards are mixed in varying but defined ratios and analyzed in replicate by LC/MS. Afterwards, the flyability ratios for these synthetic peptide pairs were calculated by using the defined mixture ratio and measured ion currents. The use of synthetic standards, quantified according to the state of the art, still includes the aberrations of the method used in the quantification of the standards and bears significant deviations in the determination in the flyability.
Summing up the above, there remains a need for improvements in methods for determining the exact ratio of distinct target-proteins, -peptides, -DNA or RNA. However these methods imply purchasing a standard with an exact ratio of standard proteins, peptides, RNA or DNA. No standards with these properties are currently available.